A step forwards in immunomodulation and immunetolerance knowledge. HLA-G expression in co-cultures of peripheral blood mononuclear cells and stem cells after in vitro NGAL stimulation

NGAL (Neutrophil Gelatinase-associated Lipocalin ) is a protein of lipocalin superfamily. Recent literature focused on its biomarkers function in several pathological condition (acute and chronic kidney damage, autoimmune disease, malignancy). NGAL biological role is not well elucidated. Several are the demonstration of its bacteriostatic role. Recent papers have indeed highlight NGAL role in NFkB modulation. The aim of this study is to understand whether NGAL may exert a role in the activation (modulation) of T cell response through the regulation of HLA-G complex, a mediator of tolerance. From 8 healthy donors we obtained peripheral blood mononuclear cells (PBMCs) and we isolated by centrifugation on a Ficoll gradient. Cells were then treated with four concentrations of NGAL (40-320 ng/ml) with or without iron. We performed flow cytometry analysis and ELISA test. NGAL increased the HLA-G expression on CD4+ T cells, with an increasing corresponding to the dose. Iron effect is not of unique interpretation. NGAL adiction affects regulatory T cells increasing in vitro expansion of CD4+ CD25+ FoxP3+ cells. Neutralizing antibody against NGAL decreased HLA-G expression and reduced significantly CD4+ CD25+ FoxP3+ cells percentage. In conclusion, we provided in vitro evidence of NGAL involvement in cellular immunity. The potential role of NGAL as an immunomodulatory molecule has been evaluated: it has been shown that NGAL plays a pivotal role in the induction of immune tolerance up regulating HLA-G and T regulatory cells expression in healthy donors. As potential future scenario we highlight the in vivo role of NGAL in immunology and immunomodulation, and its possible relationship with immunosuppressive therapy efficacy, tolerance induction in transplant patients, and/or in other immunological disorders.

Resident angiogenic mesenchymal stem cells from multiorgan donor thoracic aortas

Stem cells are one of the most fascinating areas of biology today, and since the discover of an adult population, i.e., adult Stem Cells (aSCs), they have generated much interest especially for their application potential as a source for cell based regenerative medicine and tissue engineering. aSCs have been found in different tissues including bone marrow, skin, intestine, central nervous system, where they reside in a special microenviroment termed “niche” which regulate the homeostasis and repair of adult tissues. The arterial wall of the blood vessels is much more plastic than ever before believed. Several animal studies have demonstrated the presence of cells with stem cell characteristics within the adult vessels. Recently, it has been also hypothesized the presence of a “vasculogenic zone” in human adult arteries in which a complete hierarchy of resident stem cells and progenitors could be niched during lifetime. Accordingly, it can be speculated that in that location resident mesenchymal stem cells (MSCs) with the ability to differentiate in smooth muscle cells, surrounding pericytes and fibroblasts are present. The present research was aimed at identifying in situ and isolating MSCs from thoracic aortas of young and healthy heart-beating multiorgan donors. Immunohistochemistry performed on fresh and frozen human thoracic aortas demonstrated the presence of the vasculogenic zone between the media and the adventitial layers in which a well preserved plexus of CD34 positive cells was found. These cells expressed intensely HLA-I antigens both before and after cryopreservation and after 4 days of organ cultures remained viable. Following these preliminary results, we succeeded to isolate mesenchymal cells from multi-organ thoracic aortas using a mechanical and enzymatic combined procedure. Cells had phenotypic characteristics of MSC i.e., CD44+, CD90+, CD105+, CD166+, CD34low, CD45- and revealed a transcript expression of stem cell markers, e.g., OCT4, c-kit, BCRP-1, IL6 and BMI-1. As previously documented using bone marrow derived MSCs, resident vascular wall MSCs were able to differentiate in vitro into endothelial cells in the presence of low-serum supplemented with VEGF-A (50 ng/ml) for 7 days. Under the condition described above, cultured cells showed an increased expression of KDR and eNOS, down-regulation of the CD133 transcript, vWF expression as documented by flow cytometry, immunofluorescence, qPCR and TEM. Moreover, matrigel assay revealed that VEGF induced cells were able to form capillary-like structures within 6 hours of seeding. In summary, these findings indicate that thoracic aortas from heart-beating, multi-organ donors are highly suitable for obtaining MSCs with the ability to differentiate in vitro into endothelial cells. Even though their differentiating potential remains to be fully established, it is believed that their angiogenic ability could be a useful property for allogenic use. These cells can be expanded rapidly, providing numbers which are adequate for therapeutic neovascularization; furthermore they can be cryostored in appropriate cell banking facilities for later use.

Cellule staminali mesenchimali umane da molteplici tessuti adulti: caratteristiche condivise e tessuto-specificità

L’attività di ricerca ha riguardato lo studio di popolazioni di cellule staminali mesenchimali umane (MSC) ottenute da molteplici tessuti adulti. Sono state investigate sorgenti di MSC alternative al midollo osseo, libere da conflitti etici, dotate di vantaggi per l’applicabilità clinica che vanno dalla elevata resa nel recupero cellulare alla tessuto-specificità. Le cellule ottenute dalle diverse sorgenti sono state caratterizzate immunofenotipicamente, commissionate mediante protocolli di induzione specifici per i diversi tipi cellulari ed analizzate con opportuni saggi istologici, immunoistochimici, di espressione genica e proteica. Esperimenti di cocoltura hanno permesso la descrizione di capacità immunomodulatorie e trofiche. - La placenta a termine risulta essere una ricca sorgente di cellule staminali mesenchimali (MSC). Dalla membrana amniotica, dal corion e dalla gelatina di Wharton del cordone ombelicale sono state ottenute MSC con potenzialità differenziative verso commissionamenti mesenchimali, con capacità immunomodulatorie e trofiche. Tali tessuti sono ampiamente disponibili, garantiscono una elevata resa nel recupero cellulare e sono liberi da conflitti etici. - Due popolazioni di cellule con caratteristiche di MSC sono state individuate nella mucosa e nella sottomucosa intestinale. Queste cellule possiedono caratteristiche di tessuto-specificità, sono dotate di attività trofiche ed immunomodulatorie che potrebbero essere vantaggiose per approcci di terapia cellulare in patologie quali le Malattie Infiammatorie Croniche Intestinali (IBD). - Popolazioni di cellule staminali con caratteristiche simili alle MSC sono state ottenute da isole pancreatiche. Tali popolazioni possiedono vantaggi di tessuto-specificità per approcci di terapia cellulare per il Diabete. - Sono stati investigati ed individuati marcatori molecolari (molecole HLA-G) correlati con il livello di attività immunomodulatoria delle MSC. La valutazione di tali marcatori potrebbere permettere di determinare l’attività immunosoppressiva a priori del trapianto, con l’obiettivo di scegliere le popolazioni di MSC più adatte per l’applicazione e di definirne il dosaggio. - E’ stato messa a punto una metodica e una strumentazione per il frazionamento di cellule staminali in Campo Flusso in assenza di marcatura (NEEGA-DF). Questa metodica permette di discriminare sottopopolazioni cellulari in base a caratteristiche biofisiche.

Celiachia e difetti dello smalto in età evolutiva: correlazione tra periodo di esposizione al glutine, forme cliniche di celiachia e aplotipo HLA

The association between celiac disease (CD) and dental enamel defects (DED) is well known. AIM: This study was designed to investigate the prevalence of DED in CD children and to specifically find a possible correlation between DED and gluten exposure period, CD clinical forms, HLA class II haplotype. MATERIALS AND METHODS: This study was designed as a matched case-control study: 374 children were enrolled (187 celiac and 187 non celiac). Data about age at CD diagnosis, CD clinical form and HLA haplotype were recorded. RESULTS: DED were detected in 87 celiac subject while no dental lesions were found in the remaining 100 patients; in 187 healthy controls enamel lesion were significantly less frequent (5.3 % versus 46.5% ; p<0.005).We found a correlation between DED and gluten exposure period, since among CD patients the mean age at CD diagnosis was significantly (p= 0.0004) higher in the group with DED (3.41± 1.27) than without DED (1.26± 0.7). DED resulted more frequent in atypical and silent forms than in the typical one. The presence of HLA DR 52-53 and DQ7 antigens significantly increased the risk of DED (p=0.0017). CONCLUSIONS: Our results confirmed a possible correlation between CD clinical form, age at CD diagnosis, HLA antigens and DED. The origin of DED in CD children is due to multifactorial events and further studies are needed to investigate other determinants.